Research Plan: Somatic mutations in hematopoietic progenitor cells (HPCs) drive the development of chronic blood disorders, such as CALR mutations in myeloproliferative neoplasms (MPN). In human, CALR mutations can lead to diverging clinical presentations of essential thrombocythemia (ET) or myelofibrosis (MF). Nevertheless, the study of the mutation impact on human hematopoiesis is often challenged by the admixture of normal hematopoietic cells with the neoplastic cells that cannot be distinguished by cell surface markers. To overcome this limitation, we developed a novel single-cell Genotyping of Transcriptomes (GoT) platform that directly links somatic genotypes with transcriptional profiles of thousands of single cells. Thus, GoT enabled the comparison of mutant and wildtype cells within the same sample in the context of progenitor identities. GoT was applied to CD34+ progenitor cells from patients with CALR-mutated ET and MF, revealing that transcriptional impact of CALR mutations is highly variable as a function of progenitor identity. This raises the question of what are the factors that lead to the cell identity-dependent transcriptional dysregulation by the CALR mutations? First, we will test the hypothesis that the cell?s epigenome constrains the CALR mutation-induced transcriptional impact and is, in turn, modified by the downstream effects of the CALR mutations (Aim 1). Novel single-cell multi-omics that integrates the epigenome, transcriptome and mutation status will be applied to the progenitors from primary human samples. Second, we hypothesize that the immune microenvironment may directly interact with the mutant HPCs to further modify the somatic mutation impact (Aim 2). Thus, the progenitor-to-immune cell interactions will be defined. We will interrogate the progenitors? epigenome and interactions with the immune microenvironment in ET vs. MF, to determine the distinct molecular factors that may underlie the development of marrow fibrosis (Aims 1 and 2). Third, we will examine the impact of interferon in CALR-mutated ET to define its perturbations on the epigenome and transcriptome of the mutant progenitor cells and their interactions with the immune cells (Aim 3). We will examine serial samples from patients treated with interferon in the context of their clinical response, to determine the molecular underpinnings of differential therapeutic response. Thus, we will define cell-intrinsic determinants (e.g. cell?s epigenome) and cell-extrinsic factors (e.g. immune microenvironment and therapy) that interact with somatic mutation impact to result in distinct cell identity- dependent transcriptional effects and, ultimately, distinct clinical presentations and response to therapy. Career Development Plan: Dr. Nam has outlined a 5-year career development plan to meet her goal of becoming an independent investigator in hematopoiesis research. She has organized an advisory committee composed not only of leaders in the field but also of those able to directly impact her career advancement. Finally, Weill Cornell Medicine/New York Genome Center constitute the ideal environment for attaining her scientific and career goals, given its outstanding research community and excellent track record of training independent physician-scientists.